Regarding a huge number of genes derived from a great deal of genomic sequence information of organisms, a research called “Structural Genomics” is now in progress, which is a systematic and comprehensive analysis of the relationship between structure and function of proteins by determining three dimensional structures of the proteins encoded by the respective genes. As the total genome sequences were clarified, it was found that three-dimensional protein structures considered to be innumerable actually comprise combinations of one to several thousands of basic structures (folds) and these combinations seem likely to realize the diversity of functions. Accordingly, high-throughput technologies throughout the processes from synthesis to structural analysis of proteins will make it possible to reveal all of the basic structures of the proteins and based on the knowledge of the basic structures, it will be enabled to elucidate the relationship between structure and function of the proteins.
As a system for expressing and preparing a number of protein samples at good efficiency, cell-free protein synthesis systems have been improved by various modifications such as introduction of dialysis, to obtain proteins in the order of milligrams within several hours (refer to Kigawa et al., FEBS Lett., vol. 442, pp. 15–19, 1999 and Japanese Patent Kokai publication JP-A-2000-175695). To express proteins in this cell-free protein synthesis system at good efficiency, a double-stranded DNA containing an appropriate expression regulatory region and a gene sequence coding for a target protein to be expressed is needed as a template DNA. In order to express any genes cloned in a cloning vector free of an appropriate expression regulatory sequence in a cell-free protein synthesis system, it is necessary to add an appropriate expression regulatory sequence to these genes. Therefore, several methods have been so far performed, in which a desired DNA fragment is excised from a plasmid vector comprising a gene by restriction enzymes or PCR amplification, then re-cloned in a vector having an appropriate expression regulatory sequence, or a desired DNA fragment was further amplified by PCR.
To obtain a highly efficient protein expression, it is required to promote the transcription of the gene with strong promoter or terminator sequence as well as to enhance the translation by improving the affinity between mRNA and ribosomes. Further, for quickly purifying or detecting synthesized proteins, it is also required to design fusion proteins having incorporated therein a tag sequence for affinity purification or detection.
When the template DNA suitable for such protein synthesis is prepared, however, much expense in time and effort is necessary to clone the DNA by genetic engineering techniques using living cells such as E. coli, because the methods are complicated and difficult to make high-throughput by automation. It is also problems to construct the template DNA by complex recombination, or to synthesize many kinds of primers for PCR for the optimization of respective genes.